The goal of this proposal is to explain how diverse innate immune response pathways can be activated by a single microbial product: bacterial lipopolysaccharide (LPS). While TLR4 is widely recognized to control transcriptional responses to LPS, TLR4-independent responses exist. Examples of such responses include endocytosis, activation of the transcription factor NFAT, and activation of non-canonical inflammasomes by caspase-11. The relationship between these diverse LPS responses is largely unknown, but as we will describe, at least some TLR4-independent responses control the classically-defined TLR4-dependent responses to LPS. Understanding how microbial products activate distinct signal transduction pathways may permit the design of drugs or vaccines that can target a subset of pathways therapeutically. Our proposal is founded on our recent discovery that LPS-induces the endocytosis of TLR4 by a process that does not require a functional TLR4 signaling (TIR) domain, or any of its associated adaptor proteins. Rather TLR4 is cargo for an endocytosis pathway that is activated by the LPS-binding protein CD14. While TLR4 does not direct its own endocytosis, this process is essential for TLR4 to induce TRIF-dependent signal transduction from endosomes. This discovery provides an example of how TLR4-dependent and -independent responses to LPS can be interdependent. In this grant application, we propose to 1) determine if a common signaling pathway is activated by CD14 to elicit diverse cell type-specific innate immune responses, 2) determine if common or distinct protein complexes exist that regulate diverse LPS inducible signaling pathways, and 3) identify the cell populations that directly respond to LPS produced by pathogenic bacterial infections in animal models of infection. Collectively, this work will provide important insight into the means by which diverse cellular responses to LPS are interconnected.